The present invention relates to a method for significantly increasing lentiviral production by inhibiting or preventing Heme Oxygenase 2 (HO-2) from binding to the group-specific antigen (Gag) of the viral proteins, thus allowing delivery of the viral proteins to plasma membranes for increasing viral particle maturation and production.
A lentivirus is a retrovirus with the ability to deliver significant quantities of viral RNA for integration of a DNA copy of that RNA into the host genome, even into non-dividing cells, making it one of the most efficient vehicles for gene delivery. These and other properties make lentiviruses of particular importance to biotechnology and pharmaceutical industries, and efforts are underway to develop RNA interference technology, gene editing, and long-term stable expression of exogenous genes from lentiviruses.
For example, lentiviruses have proven particularly useful for gene therapies targeting the central nervous and hematopoietic systems (Ginn S L, Alexander I E, Edelstein M L, Abedi M R, Wixon J. Gene therapy clinical trials worldwide to 2012—an update. J Gene Med. 2013 February; 15(2):65-77). Also, lentiviruses have been used for RNA interference, genetic editing, and stable gene expression purposes, by successfully delivering ZFNs, CRISPR/Cas9, luciferases, shRNA, IncRNAs and more (Ginn S L et al, supra; Giacca M, Zacchigna S. Virus-mediated gene delivery for human gene therapy. J Control Release. 2012 Jul. 20; 161(2):377-88; Ausubel L, Couture L, et al. Production of CGMP-Grade Lentiviral Vectors. Bioprocess Int. 2012 February; 10(2): 32-43; and Negre O, et al., Gene Therapy of the β-Hemoglobinopathies by Lentiviral Transfer of the β(A(T87Q))-Globin Gene, Hum Gene Ther. 2016 February; 27(2):148-65. doi: 10.1089/hum.2016.007). By 2012, more than 1,800 gene therapy clinical trials had been undertaken with viruses representing at least 66.8% of all vectors used (Ginn S L et al, supra). Thus, lentiviruses have been successful vectors for the treatment of genetic disease in humans, measurable brain disease, and hematopoietic stem cell therapy (Lenti-Globins). Also, lentiviruses can deliver nucleic acids to a range of host cell lines including mammalian and non-dividing cells (Ginn S L et al, supra; Giacca M et al.). But the ongoing challenge facing commercial and large-volume production of lentiviruses, especially for phase I & II clinical trials, is the inconsistent and low titers (Ausubel L et al. supra).
A major factor limiting the broad application of lentiviruses for these and other purposes is the time and cost required to produce large quantities of viral particles collected from cell lines that can express and synthesize structural proteins for harvesting. N-myristoylation is the covalent attachment of myristic acid, the 14-carbon saturated fatty acid, to the N-terminal glycine of proteins in eukaryotic cells. A large number of proteins of diverse functions are modified by N-myristoylation (Thinon et al., 2014). The addition is catalyzed by N-myristoyltransferases (NMTs), and two isoforms (NMT1 and NMT2) encoded by distinct genes have been identified in mammalian cells (Boutin, 1997; Giang and Cravatt, 1998). Myristoylation is generally permanent and irreversible. NMT1 homozygous knockout mice are not viable, indicating that myristoylation is essential for development (Yang et al., 2005). Myristoylated proteins are involved in a wide variety of physiological activities such as virus replication, cell signaling pathways, oncogenesis, and apoptosis [for review, see (Wright et al., 2010)]. Examples of myristoylated proteins include the retrovirus Gag structural protein (Henderson et al., 1983), tyrosine kinase Src and Src kinase family members (Cross et al., 1984), phosphatases such as calcineurin B (Aitken et al., 1982), the BH3 domain protein BID (a key mediator of apoptosis) (Zha et al., 2000), and TRAM (Toll-like receptor adaptor molecule, aka TICAM2), a mediator of TLR4 signaling (Rowe et al., 2006). Many, but not all, myristoylated proteins reside in intracellular membranes.
The Gag and Gag-Pol precursor proteins of nearly all retroviruses are modified by the cotranslational addition of myristate to the amino-terminal glycine of the matrix domain (MA) (Gottlinger et al., 1989; Henderson et al., 1983; Palmiter et al., 1978). The avian alpharetroviruses are exceptions to the rule, and instead their Gag and Gag-Pol proteins are modified by N-terminal acetylation. The N-myristoylation of all other Gags is essential for replication of these retroviruses, and inhibition of the NMT's enzymatic activity or mutation of the Gag N-terminal glycine to alanine to prevent myristoylation blocks the spread of virus in host cells (Bryant and Ratner, 1990; Gottlinger et al., 1989; Rein et al., 1986). When Gag myristoylation is prevented, the Gag protein remains in the cytoplasm and is not properly delivered to the plasma membrane for virion assembly and budding (Bryant and Ratner, 1990; Ono and Freed, 1999). Mutational studies have revealed that the N-terminal myristate, and also a cluster of basic amino acids constituting a small basic patch on the surface of MA, are both required for membrane binding of Gag (Resh, 2005). The basic residues of Gag are thought to interact with the negatively charged phospholipids of the plasma membrane to promote its membrane association (Hill et al., 1996). It has been proposed that in the cytoplasm the N-terminal myristate of Gag is initially trapped by a hydrophobic pocket in the MA domain, limiting the interaction between Gag and endogenous membranes, and conformational changes (a “myristoyl switch”) associated with virus maturation expose the myristate (Hermida-Matsumoto and Resh, 1999; Resh, 2004). The plasma membrane-specific lipid PI(4,5)P2 can compete with myristate for binding to the hydrophobic pocket, promoting the exposure and insertion of the myristate tail into the plasma membrane and thus facilitating virus budding (Bouamr et al., 2003; Saad et al., 2007; Zhou and Resh, 1996). The bulk of the MA domain is not absolutely required for membrane association and virion budding. An HIV-1 Gag mutant lacking most of MA and a portion of CA, but retaining the N-terminal myristoylation (so-called “miniGag”) can efficiently mediate virion assembly and release (Accola et al., 2000; Reil et al., 1998), suggesting that the exposure and insertion of the myristate tail is the primary determinant for the membrane association of Gag and virus budding.
It has long been supposed that there must be proteins that bind myristoylated substrates and regulate their localization and function, but few have been identified. UNC119 is a lipid-binding protein of photoreceptors (Higashide and Inana, 1999; Swanson et al., 1998) that interacts with acylated rod photoreceptor transducin α subunit (Tα) and myristoylated ciliopathy protein nephrocystin-3 (NPHP3) (Constantine et al., 2012; Wright et al., 2011; Zhang et al., 2011). An early search revealed a protein of 32 kDa that bound to a myristoylated v-Src peptide (Resh and Ling, 1990), but its identity has not previously been established.
The present invention now has discovered how to increase the production of large quantities of lentivirus particles that are assembled from proteins that have been myristoylated or that carry other acyl chains of fatty acids. The method thus addresses the need for such increased production.